US20090264510A1 - Double helical oligonucleotides interfering with mRNA used as effective anticancer agents - Google Patents

Double helical oligonucleotides interfering with mRNA used as effective anticancer agents Download PDF

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US20090264510A1
US20090264510A1 US12/221,063 US22106308A US2009264510A1 US 20090264510 A1 US20090264510 A1 US 20090264510A1 US 22106308 A US22106308 A US 22106308A US 2009264510 A1 US2009264510 A1 US 2009264510A1
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oligonucleotides
sirna
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Maciej Wieczorek
Joanna Wietrzyk
Anna Nasulewicz
Katarzyna Szczaurska
Jan Piotr Guzenda
Monika Lamparska-Przyhysz
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CELON PHARMA SP Z O O
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
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    • C12N2330/30—Production chemically synthesised

Definitions

  • the teachings of all of the references, including websites, cited herein are incorporated herein in their entirety by reference.
  • the present invention relates to the application of double-stranded oligonucleotides interfering with the mRNA transcribed from a gene involved in carcinogenesis, particularly the Wnt1, Wnt2 or Her3 gene, as novel anti-tumour agents.
  • RNA interference is a phenomenon based on the post-transcriptional gene silencing (PTGS) and is an excellent tool for the analysis of their function and role in many processes within an organism. This technique is of great importance in functional genomics, mapping of biochemical pathways, determination of pharmacological treatment directions and in gene therapy.
  • PTGS was first described in plants (Napoli, C., C. Lemieux and R. Jorgensen. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co - Suppression of Homologous Genes in trans. Plant Cell 2:279-289, 1990) In 1998, Andrew Fire and Craig Mello described RNAi for the first time in an animal, C. elegans (Fire, A.
  • nucleotides this long also elicited an immune response (increased interferon levels) in mammalian cells and it was T. Tuschl, S M. Elbashir et al. who finally discovered that the application of short, double-stranded nucleotides (19-21 bp) does not induce an immune response (Elbashir, S. M., J Harborth, W. Lendeckel, A. Yalcin, K Weber and T. Tuschl. 2001 . Duplexes of 21- nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498).
  • RNAi double-stranded interfering nucleic, acid (iNA) preferably a double-stranded RNA (dsRNA) molecules, also called siRNA.
  • RNAi is a response to cellular processes induced by dsRNA, which degrades homologous mRNA. Even a few copies of dsRNA may entirely destroy the transcripts for a given gene formed within a cell. The destruction of selected mRNA's through RNAi begins with the activation of RNAse III, which cleaves long hairpin loops of dsRNA or ssRNA fragments into double-stranded small interfering RNA (siRNA) 21-23 nucleotides long. siRNA's prepared earlier may be introduced into cells externally.
  • siRNA molecules bind to a nuclease complex forming a RISC(RNA induced silencing complex). Thanks to the helicase activity which is a part of the RISC, dsRNA is separated into single strands. The ssRNA molecules formed then anneal to complementary mRNA strands. The final stage of PTGS is the degradation of selected mRNA by RISC nucleases. In contrast to traditional methods, such as knockouts, gene silencing is quickly and easily performed, both in animal and in cell line models.
  • the authors of the present invention have performed intensive research and have determined that the silencing of expression of genes involved in carcinogenesis, e.g. gene Wnt1, using double-stranded oligonucleotides such as an iNA or an siRNA for this gene is an effective strategy for the inhibition of tumour cell proliferation.
  • genes involved in carcinogenesis e.g. gene Wnt1
  • double-stranded oligonucleotides such as an iNA or an siRNA for this gene
  • Wnt1 is a secretory protein which binds the “frizzled” inter-membrane receptor and transmits a signal to cytoplasmatic phosphoproteins, which in turn downregulate the constitutively high activity of glycogen synthase kinase 3Beta (GSK-3Beta) (Polakis et al., Wnt signaling and cancer, Genes Dev. 2000 Aug. 1; 14(15):1837-51). The result of this is the stabilization and growth of Beta-catenin levels in the cell nucleus.
  • GSK-3Beta glycogen synthase kinase 3Beta
  • Wnt-1 overexpression has been noted in many types of tumours, including in cancers of the lung, colon and breast, sarcomas and tumours of the head and neck (Katoh et al. Expression and regulation of WNT 1 in human cancer: up - regulation of WNT 1 by beta - estradiol in MCF -7, In J Oncol, 2003 January; 22(1):209-12).
  • Anti-WNT-1 monoclonal antibodies are known.
  • the application of such antibodies resulted in an increase of apoptosis, a decrease in tumour cell proliferation (H460 and MCF-7 lines), as well as inhibition of the take of transplantable murine lung cancer (H460) (Biao He, A Monoclonal Antibody against Wnt -1 Induces Apoptosis in Human Cancer Cells, Neoplasia, Vol. 6, No. 1, January/February 2004, pp. 7-14).
  • Biao He et al. also used chemically unmodified siRNA on a breast cancer line (MCF-7), resulting in an increased apoptosis rate in these cells.
  • Anti-WNT-1 monoclonal antibodies elicited apoptosis in sarcoma cells (A-204) (Iwao Mikami, Efficacy of Wnt -1 monoclonal antibody in sarcoma cells, BMC Cancer 2005, 5:53, 24 May 2005), and in NCI-H1703 and H28 lung cancer cells (Liang You, Inhibition of Wnt -1 Signaling Induces Apoptosis in ⁇ - Catenin - Deficient Mesothelioma Cells, Cancer Research 64, 3474-3478, May 15, 2004).
  • This research also made use of chemically unmodified siRNA in MCF-7 breast cancer cells, and NCI-H1703 and H28 lung cancer cells, resulting in an increased apoptosis rate.
  • liver cancer line cells expressing type C hepatitis virus core protein.
  • Expression of type C hepatitis virus core protein was obtained through the transfection of these cells with vectors coding for said protein.
  • the presence of this protein enhanced WNT-1 expression and cell proliferation.
  • the application of siRNA specific for Wnt-1 in such cells caused the silencing of its expression and inhibited proliferation.
  • This sort of experimental model does not provide evidence which would allow one to hypothesize that a similar effect would be elicited in cells unmodified with the viral protein, upon the application of WNT-1 specific siRNA.
  • the present invention relates to the use siRNA against mRNA transcribed by genes involved in carcinogenesis.
  • An example of such an siRNA is one that targets the mRNA of the oncogene Wnt1 gene.
  • An siRNA directed to tumour cell lines expressing the Wnt1 results in a strong inhibition of tumour cell proliferation. This inhibition is dose-dependent.
  • the present invention thus successfully delivers a solution to the problem of tumour treatment through the inhibition of tumour cell growth, using the RNA interference mechanism to degrade the mRNA of the gene involved in carcinogenesis, e.g. gene coding WNT-1.
  • the present invention provides methods of inducing apoptosis or inhibiting growth of a cancer cell as well the method for obtaining the oligonucleotide useful as an effective anticancer agent. Furthermore, the application of the present invention entails a very limited danger of eliciting an immune response in treated patients.
  • the production of double-helical oligonucleotides is a reproducible process and is simple to perform using standard equipment, the so-called RNA synthesizers.
  • Such oligonucleotides may be designed according to one of many algorithms described to date, such as the one indicated in Example 1.
  • the sequence of an mRNA gene of interest can be obtained from a database, for example GenBank, and the NCBI Reference Sequence should be chosen.
  • the second structure of the mRNA target sequence can be designed using computer folding algorithm.
  • siRNAs against a chosen mRNA sequence can be generated in silico using known algorithms. There are many algorithms available on-line that are used to design siRNAs against a particular mRNA sequence. These algorithms in general are based on similar equations but there are subtle differences among them. Most of algorithms are based on Tuschl rules of siRNA design but some of them additionally use also Reynolds rules (Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W S, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 March; 22(3):326-330). It is known that you have to verify siRNAs generated by one of the algorithms with another algorithm. That is why in our method we use different algorithms based on different equations.
  • the Tuschl Rules are:
  • Target sequence from a given cDNA sequence beginning 50-100 nt downstream of start condon 2. First search for 23-nt sequence motif AA(N 19 ). If no suitable sequence is found, then, 3. Search for 23-nt sequence motif NA(N 21 ) and convert the 3′ end of the sense siRNA to TT 4. Or search for NAR(N 17 )YNN 5. Target sequence should have a GC content of around 50%, wherein
  • Some algorithms also analyze the thermodynamic stability of siRNAs.
  • the distribution of free energy through siRNA molecule is a very important factor in describing the potential of a given sequence. This feature is very important in recognition of the guide strand by RISC because RISC recognizes the 5′ end of a strand that will be incorporated into RISC, and will serve as the guide strand. It is known that the 5′ end of the antisense strand should be less stable than the 3′ end, so the free energy at the 5′ end should be higher than at the 3′ end.
  • GC content should be between 30%-60%; this will ensure that a designed duplex will not be too stable to be unwound and will be stable enough to avoid self-unwinding in cytoplasm.
  • siRNA In thermodynamics analysis it is also recommended to design siRNA with a low stability at position 10 of antisense strand. This position is a cleavage site so there should not be formed a strong duplex between guide strand and target mRNA, U base is recommended in this position.
  • Another factor that should be taken into consideration during siRNA designing is to target secondary structure accessibility. This factor describes probability of a single stranded motif in target region in mRNA molecule. In the cytoplasm mRNA never exists as a single strand, its secondary structure is rich in hairpins, loops and other structures which are results of partial paring between bases in given mRNA molecule.
  • siRNA is designed to avoid potential “off-target” effect. This effect occurs if particular siRNA targets not only desired mRNA but other mRNAs as well. In this case there are also many algorithms like blast or clustal which can predict possible interactions with any known transcript.
  • the sequence of an mRNA gene of interest was obtained from a database, for example GenBank, and the NCBI Reference Sequence was chosen. siRNAs against chosen mRNA sequence were generated in silico using known algorithms.
  • the designed sequences were ranked according to total filtering score based on following rules:
  • sequences were ranked by the inhibition score.
  • sequences were ranked by the decrease in mRNA level score.
  • sequences were ranked by the decrease in protein level score.
  • siRNAs with z factor better or equal to 50% of the best sequence, but not more than 3 were analyzed according to the cell death mechanism. Sequences were ranked by:
  • the oligonucleotides be no more than 30 bp long, and preferentially be 21-23 bp long.
  • Sense and antisense oligonucleotides may be symmetrical or not, meaning that i.e. 2 terminal nucleotides may be unhybridized, thus forming sticky ends.
  • the oligonucleotides may be modified chemically. Chemical modifications may pertain to phosphates, ribose or the nucleases themselves. Said chemical modifications may pertain to only selected nucleotides, i.e. terminal or median, or the entire oligonucleotide.
  • the oligonucleotides may be delivered to tumour cells both by themselves, without vectors, as well as with a vector, both viral and non-viral.
  • Adenoviruses or adeno-like viruses are examples of viral vectors, which facilitate the continual expression of the oligonucleotide following introduction into tumour cells.
  • Non-viral vectors used to introduce oligonucleotides into cells are lipid capsules, lipid complexes or other vectors prolonging their half-lives in a living organism and/or absorption into cells. As a result of use of present invention, considerable inhibition of tumour cell proliferation is achieved.
  • FIG. 1 Percent of proliferation inhibition after transfection of MCF-7 cells with sixteen siRNAs sequences against Wnt1 gene in concentration 50 nM for 48 h with respect to untreated cells. Cells viability was measured using MTS test.
  • FIG. 2 Decrease of Wnt1 protein level in MCF-7 cells after transfection with specific siRNA to Wnt1.
  • FIG. 3 Cell cycle analysis after treatment with siRNA against Wnt1. a) Cytograms showing DNA content and cell size of MCF-7 cells 72 h after transfection with W15 and WP sequences. b) Histograms presenting cell cycle of MCF-7 cells 72 h after transfection with W15 and WP sequences.
  • FIG. 4 Apoptosis after treatment with W15 sequence. a) Activity of caspases 3 and 7. b) Morphological changes of MCF-7 cells after treatment with W15 sequence.
  • FIG. 5 Apoptosis analysis using Annexin V and propidium iodide staining of MCF-7 cells after Wnt1 siRNA. a) Cytograms presenting morphology of MCF-7 cells 72 h after transfection with W15 and WP sequences. b) Cytograms showing number of cells in early, late phase of apoptosis and necrosis 72 h after transfection with W15 and WP sequences.
  • FIG. 6 Wnt1 siRNA induces apoptosis triggered by decrease in protein level of Wnt1 in MCF-7 cells.
  • FIG. 7 siRNAs ranking results. Eight siRNAs passed inhibition score ranking (bold), two sequences passed z score ranking (bold, red). All factors for sequence WP (sequence from literature) for comparison were analyzed.
  • Human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (Rockville, Md., USA). Cell cultures were maintained in DMEM supplemented with 10% (v/v) fetal calf serum (FCS), 50 ⁇ g/ml gentamycin, 2.5 ⁇ g/mL fungizone, 50 UI/mL penicillin and 50 ⁇ g/ml streptomycin (Invitrogen Carlsbad, Calif. USA) in an atmosphere of 5% CO2/95% humidified air at 37° C., and routinely subcultured every 2 or 3 days.
  • FCS fetal calf serum
  • MCF-7 cells were plated in Opti-MEM (Invitrogen) at 7 ⁇ 103 cells per well in 96-well plates one day before experiments. The next day the MCF-7 cells were transfected with fifteen siRNAs sequences specific to Wnt1 mRNA and scrambled siRNA sequence (control) in at a concentration of 50 nM for 48 h using Lipofectamine RNAi MAX (Invitrogen) according to manufacturer's protocol. siCONTROL TOX (Dharmacon, Chicago, Ill. USA) was used as a control of transfection efficiency. After 48 h of experiment proliferation inhibition was measured using MTS test (Promega, Madison, Wis. USA).
  • Reagents for Western blotting were purchased from BioRad (Hercules, Calif. USA), anti-Wnt1 antibody was from Zymed Invitrogen, anti-actin, anti-phosphor-beta-catenin, anti-c-myc and anti-cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, Calif. USA), anti-cleaved PARP antibody was from Cell Signaling (Danvers, Mass., USA).
  • Western blotting detection reagents was from Roche Diagnostics (Indianapolis, Ind., USA) and Light Film BioMax was from Kodak (Rochester, N.Y. USA)
  • the supernatant was removed and the cell pellet was re-suspended in 0.5 mL Total Lysis Buffer RIPA (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), and incubated at 4° C. for 30 min.
  • the cells suspended in the buffer were centrifuged at 9000 g, 10 min, at 4° C., then the supernatant (containing the total protein fraction) was carefully removed and passed six times through a 20-gauge syringe needle.
  • the lysates were mixed 1:2 (v/v) with Laemmli sample buffer (BioRad) containing 2.5% 2-mercaptoethanol and boiled for 3 min. Samples containing identical quantities of proteins were subjected to SDS-PAGE (12% gel) together with a Kaleidoscope Marker (BioRad). The electrophoresis was run for 1 hour at 100 V using a Mini Protean III cell (BioRad,). After electrophoresis the separated proteins were electroblotted on a PVDF membrane (Biorad) for 70 min at 110 V using the Mini Protean III. The membranes were blocked overnight with 5% w/v solution of non-fat powdered milk in TBST (pH 7.5).
  • the membranes were rinsed three times for 10 min in TBST, at room temperature, and then incubated for 1 hour at room temperature with the primary antibodies diluted 1:200.
  • the membranes were then rinsed four times for 10 min in TBST and incubated with diluted 1:2000 secondary antibodies conjugated with horseradish peroxidase (Sigma Aldrich, St. Louis, USA) for another 1 h at room temperature.
  • the membranes were rinsed three times for 10 min in TBST, and labelled proteins were visualized using the LumiLight (Roche) Western blotting detection reagent on a high performance chemiluminescence BioMAX light film (Kodak). The image on light film was then analyzed with a Kodak Edas System and the integrated optical density (IOD) was measured.
  • the cells were then fixed in 1% formaldehyde for 15 min, washed twice with PBS, suspended in ice-cold 70% ethanol and stored at ⁇ 20° C. for 24 h. after this time the cells were washed twice with PBS-1% BSA and incubated for 1 h with either primary antibody anti-Wnt1 (Zymed-Invitrogen) diluted 1:250 with PBS-1% bovine serum albumin (BSA). After primary incubation the cells were washed twice with PBS-1% BSA, and incubated for 1 h with 1:500 secondary antibodies labelled with Alexa Fluor 488 (Molecular Probes, Eugene, Oreg., USA).
  • primary antibody anti-Wnt1 Zymed-Invitrogen
  • BSA bovine serum albumin
  • the cells were then washed twice in PBS-1% BSA and finally incubated with a 10 ⁇ g/mL solution propidium iodide with RNase A for 15 min to counterstain the DNA. Then the cells were measured using BD FACS Calibur Flow Cytometry (Becton Dickinson, Franklin Lake, N.J., USA)
  • MCF-7 cells were plated in Opti-MEM (Invitrogen) at 7 ⁇ 103 cells per well in 96-well plates one day before experiments. On the next day the MCF-7 cells were transfected with siRNAs sequences which passed the inhibition score ranking at a concentration of 50 nM for 48 h using Lipofectamine RNAi MAX (Invitrogen) according to manufacturer's protocol. After 12 h of siRNA exhibition, activation of caspases 3 and 7 was measured using Caspase-Glo 3/7 assay (Promega) by GloMaxTM 96 Microplate Luminometer (Promega) according to manufacturer's protocol.
  • apoptosis cells transfected with siRNA which passed the final screening test were harvested by trypsinization and stained using an Annexin V FLUOS Staining Kit (Roche Diagnostics, Indianapolis, Ind., USA), according to the manufacture's protocol. Then stained cells were immediately analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lake, N.J.). Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to Annexin V-FITC but excluded propidium iodide. Cells in necrotic or late apoptotic stages were labeled with both Annexin V-FITC and propidium iodide.
  • RNA synthesis was performed using the solid phase synthesis technique, using typical protocols for the synthesis of nucleic acids using derivatives of ⁇ -cyanoethyl phosphamide esters in conjunction the tert-butyldimethyl-silane protection of the 2′-OH group of ribose.
  • Phsosphamide monomers attach to the free 5′-OH group of ribose following activation with 5-benzylmercapto-1H-tetrazole. This reaction proceeds rapidly, efficiently yielding oligomers.
  • the oligomers formed are additionally purified using chromatographic (HPLC) or electrophoretic (PAGE) techniques. The synthesis was performed on an Applied Biosystems 962 RNA synthesizer.
  • siRNA was produced through the gentle agitation of equimolar amounts of complementary RNA strands for 1 hour at ⁇ 20° C. in 2M acetate buffer in ethanol. Such a solution was centrifuged for 15 min. and dried with 70% ethanol.
  • SiRNA (WNT1 — 16) was diluted using the Hiperfect lipid vector.
  • Hiperfect was purchased from and supplied by Qiagen. Each siRNA dilution was prepared in a series and then an appropriate amount of HiPerFect was added. 25 nM 3 ⁇ l siRNA+1197 ⁇ l medium without serum
  • Example 3 The dilutions prepared in Example 3 were used in transfection. siRNA transfection was performed at three concentrations: 1, 5 and 25 nm, HiPerfect: constant 0.75 microL/well. Experimental controls consisted of: a) tumour cells, b) a+HiPerfect reagent
  • the “Blank” control consisted of a solution from wells containing only culture medium.
  • the positive control consisted of cells suspended in culture medium.
  • the spectrophotometrically determined OD is proportional to the number of living cells in a sample.
  • the results obtained from the measurement of the proliferation rate of individual tumour line cells treated with siRNA were collected in tables (Table 2 and Table 3)
  • Non-coding siRNA had almost no effect on cell proliferation and transfection efficiency in these experiment was roughly 88%. Few of tested siRNA sequences showed great ability to reduce cell proliferation, in some cases over 50% that means higher than cytostatic drug (Docetaxel). The sequence that reached the best results on proliferation rate was W15 which inhibited proliferation by 75% relative to untreated cells and was much more effective than docetaxel and WP siRNA known from literature (He et al. 2004).
  • siRNA is Specific and Potent in Decreasing Level of Wnt1 mRNA
  • MCF-7 cells transfected with siRNA against Wnt1 mRNA would cause a decrease in mRNA level. Analysis was performed 48 h after transfection. Total mRNA isolation, transcription to cDNA and real-time PCR were done as described in Material and Methods. After MCF-7 cells were transfected with an siRNA sequence that targets W15 mRNA, we observed a decrease in mRNA by 61% in comparison to untreated control cells. This experiment was also a control indicating the specificity of our sequence. Additionally we performed a similar experiment with A549 cells, to check if there would be any response.
  • siRNA Specific to Wnt1 mRNA Decreases Protein Level
  • FIG. 2 a Western blotting analysis of Wnt1 level in MCF-7 cells after transfection with siRNA against Wnt1 was done ( FIG. 2 a ). There was a decrease of Wnt1 level in cells treated with siRNA that targets the W15 mRNA sequence after 48 h and to lesser extent but also of significance in cells treated with siRNA that targets the W13 mRNA sequence relative to the control. There was a slight decrease of Wnt1 level after WP sequence treatment of MCF-7. Western blotting analysis showed an increase in the level of phosphorylated beta-catenin in MCF-7 cells after the cells were treated with siRNA that targets the Wnt1 mRNA. We observed a correlation between a decline of Wnt1 level and a decrease of c-myc and cyclin D1 levels in MCF-7 cells treated with W15 or W13 sequence. We did not observe such changes after WP sequence treatment.
  • W15 sequence against Wnt1 provides a decrease of Wnt1 level in MCF-7 cells, and it is correlated with a decline of c-myc, cyclin D1 and an increase of phosphorylated beta-catenin level.
  • caspases activation assay To verify what kind of cell death is triggered by siRNA treatment we performed caspases activation assay. The results obtained in this assay are presented as inhibition of proliferation in comparison to control. We observed that after treatment of MCF-7 cells with W15 sequence there was at least fivefold increase in activation of caspases 3 and 7, and after W13 sequence treatment it was around fourfold increase while after treatment with cytotoxic docetaxel it was only about twofold increase ( FIG. 4 a ). This results were confirmed by morphological changes of MCF-7 cells after treatment with W15 sequence ( FIG. 4 b ). These results show that the siRNA sequence that targets the W15 mRNA sequence induces apoptosis in MCF-7 cells.
  • AV positive and PI negative are viable cells in early phase of apoptosis, while AV positive and PI positive are cells in a late phase of apoptosis.
  • Necrotic cells are AV negative and PI positive ( FIG. 5 ).
  • Flow cytometry technique was used to verify if apoptosis was triggered by of the reduction in the level of Wnt1 in MCF-7 cells transfected with siRNA that targets the Wnt1 mRNA.
  • FIG. 6 Flow cytometry technique was used to verify if apoptosis was triggered by of the reduction in the level of Wnt1 in MCF-7 cells transfected with siRNA that targets the Wnt1 mRNA.
  • the cDNA sequence of the WNT-1 gene (Accesion No. NM005430) (SEQ ID NO: 33) 1 gcggtgccgc ccgccgtggc cgcctcagcc caccagccgg gaccgcgagc catgctgtcc 61 gccgcccgcc cccagggttg ttaaagccag actgcgaact ctcgccactg ccaccgc 121 cgcgtcccgt caccgtcg cgggcaacaa ccaaagtcgc cgcaactgca gcacagagcg 181 ggcaaagcca ggcaggccat ggggctctgg gcgctggtttgctgctacg 241 ctgct
US12/221,063 2006-01-31 2008-07-30 Double helical oligonucleotides interfering with mRNA used as effective anticancer agents Abandoned US20090264510A1 (en)

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PL378857A PL378857A1 (pl) 2006-01-31 2006-01-31 Podwójnie skręcone oligonukleotydy interferujące z mRNA genu WNT1 (siRNA) stosowane w celu hamowania poliferacji komórek nowotworowych
PCT/PL2007/000006 WO2007089161A2 (en) 2006-01-31 2007-01-31 DOUBLE HELICAL OLIGONUCLEOTIDES INTERFERING WITH mRNA USED AS EFFECTIVE ANTICANCER AGENT

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN103068980A (zh) * 2010-08-02 2013-04-24 默沙东公司 使用短干扰核酸（siNA）的RNA干扰介导的联蛋白（钙粘蛋白关联蛋白质），β1（CTNNB1）基因表达的抑制
CN114464259A (zh) * 2022-01-14 2022-05-10 郑州大学 一种靶向PD-1 mRNA反义脱氧寡核苷酸的筛选方法及应用
CN116825199A (zh) * 2023-02-21 2023-09-29 王全军 筛选siRNA序列以降低脱靶效应的方法及系统

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